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Available online at http://www.journalcra.com INTERNATIONAL JOURNAL OF CURRENT RESEARCH International Journal of Current Research Vol. 10, Issue, 08, pp.72879-72883, August, 2018
ISSN: 0975-833X RESEARCH ARTICLE
NEW ISOFLAVONOID FROM THE ROOTS OF ALLEXIS OBANENSIS (VIOLACEAE) AND EVALUATION OF ANTIBACTERIAL, ANTIOXIDANT AND ANTIPLASMODIAL ACTIVITIES
1,*Nganso Ditchou Yves Oscar, 2Ndogo Eteme Olivier, 2Zondegoumba Ernestine, 3Mala Opono M. T. G. and 2Nyasse Barthelemy
1Department of Chemistry, Faculty of Science, University of Maroua, Cameroon 2Department of Organic Chemistry, Faculty of Science, University of Yaounde I, Cameroon 3Department of Biochemistry, Faculty of Science, University of Yaounde I, Cameroon
ARTICLE INFO ABSTRACT
Article History: In the search for new compounds endowed with antibacterial, antioxidant and antiplasmodial Received 22nd May, 2018 properties in Cameroonian pharmacopoeia, a new compound was established using spectroscopic Received in revised form analysis techniques. The compound was isolated from the ethyl acetate fraction of Allexis obanensis. 14th June, 2018 The antibacterial, antioxidant and antiplasmodial activities of this compound isolated were assessed in Accepted 27th July, 2018 st this study. It showed good antibacterial activities, good antiplasmodial activity against the Published online 31 August, 2018 chloroquine-sensitive Plasmodium falciparum 3D7 strain but has presented a weak antioxidant activity. Key Words:
Antibacterial, Antioxidant, Antiplasmodial, Isoflavonoid, NMR Structure elucidation, Natural products.
Copyright © 2018, Nganso Ditchou Yves Oscar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Citation: Nganso Ditchou Yves Oscar, Ndogo Eteme Olivier, Zondegoumba Ernestine, Mala Opono M. T. G. and Nyasse Barthelemy. 2018. “New isoflavonoid from
the roots of allexis obanensis (violaceae) and evaluation of antibacterial, antioxidant and antiplasmodial activities”, International Journal of Current Research, 10, (08), 72879-72883.
INTRODUCTION 7-hydroxy-3-(3-hydroxy-4- methoxyphenyl) -5-methoxy-4H- chromen-4-one (1) which was extracted from the roots of Allexis obanensis belonging to the family Violaceae, is Allexis obanensis. We also evaluated the antibacterial, commonly distributed from Congo to Cameroon (Hutchinson, antiplasmodial and antioxidant activities of this compound. 1954). Violaceae plants have been constantly used in traditional medicine to treat many diseases caused by RESULTS AND DISCUSSION pathogenic agents. The bark of Allexis cauliflora are used to treat fever and syphilis (Achoundong, 2010; Achoundong, The ethyl acetate fraction of Allexis obanensis roots was 1998). Allexis genera have been reported as matrix purified by column chromatography on silica gel from which metalloproteinase inhibitors (Nganso et al., 2011), antioxidants we obtained one new isoflavonoid. The new isoflavonoid was (Vukics et al., 2008), stomach aches and antiplasmodials obtained in the form of a white powder in the CH2Cl2/MeOH (Moon, 2007), but experiments to isolate antiplasmodial and (50:1) solvent system. It showed the base peak of the molecular antioxidant agents from Allexis obanensis were not conducted. ion at m/z = 314.1236 (M + Na)+, (calcd. 312.10) in HRESI Despite the availability of this ethno pharmacological MS, corresponding to the formula C17H14O6Na. the UV information, there is no phytochemical report published on spectrum showed absorption bands at ƛmax 241, 248 and Allexis obanensis to date. In the present study, we report the 277nm, suggesting an isoflavonoid ring (18). The IR spectrum isolation and structural elucidation of a new compound named presented vibration bands at vmax 3324 (hydroxyl groups) 1630 and 1580 cm-1 (aromatic chain).On its 1H NMR spectrum, we observed a doublet of doublet of 1H integration at δ = 7.15 *Corresponding author: Nganso Ditchou Yves Oscar ppm (J = 8.2, 1.9 Hz) and an integration doublet 1H at δ = 6.93 Department of Chemistry, Faculty of Science, University of Maroua, Cameroon ppm (J = 1.9 Hz) attributable to H-6'and H-2' respectively. DOI: https://doi.org/10.24941/ijcr.32154.08.2018 72880 Nganso Ditchou Yves Oscar et al. New isoflavonoid from the roots of allexis obanensis (violaceae) and evaluation of antibacterial, antioxidant and antiplasmodial activities
An integration doublet 1H at δ = 6.84ppm (J = 8.2 Hz) the new isoflavonoid shows that the CMB/CMI ratio is 2 for attributable to H-5'. CM64, BM67, ATCC8739, EA289, PS299645,ATCC11296, and EA294 strains. Antioxidant activities were achieved by two These three signals indicate a disubstitution of ring B. Two complementary tests, namely the DPPH assay for radical- doublets of integration 1H each, the first at δ = 6.28 ppm (J = 2 scavenging activity and the Ferric reducing-antioxidant power Hz) and the second at δ = 6.42 ppm (J = 2 Hz) attributable to (FRAP) assay for assessing reducing power. The scavenging H-8 and H-6 respectively indicating a disubstitution of ring A activity of DPPH is considered as classic, simple, rapid and in positions 5 and 7. Two singlets of integration of 3H at δ = inexpensive method for evaluating antioxidant properties. This 3.91 and 3.9 ppm revealing the presence of two methoxyl method is based on the ability of compounds to act as H donors groups in the molecule. The analysis of the spectrum relative to to reduce free radicals. However, this method is unable to the HSQC experiment makes it possible to assign the signals of highlight the reducing power of an antioxidant compound. For the protons to the corresponding carbon atoms thus: The H- this reason, the FRAP assay was added as a complementary 6'allows the attribution of the signal at δ = 115.4 ppm to the method (Messi et al., 2016). carbon atom C-6'. H-2'allows the assignment of the signal at δ = 120.8 ppm to the carbon atom C-2'. H-5'allows the This table shows that compound 1(07.50±0.48) has presents a assignment of the signal at δ = 112.4 ppm to the C-5' carbon weakly DPPH radical scavenging activity than the standard atom. The two families of protons of the methoxyl groups reference ascorbic acid (SC50 = 4.50 mM). The reducing power carried the carbons at δ = 55.6 ppm; δ = 48.01 ppm. According assay is based on the reduction of Fe3+ in potassium to the information collected from the HMBC spectrum, the H- ferricyanide to Fe2+ which forms a blue complex. The 2'and H-6' protons show: Two correlation spots with the same formation of this blue complex is monitored at ƛ= 593 nm. The carbon atom at δ = 146.65 ppm which can only be attributed to greater the reducing power of the analyte, the greater the carbon C-4. This same carbon atom shows a correlation spot concentration of the complex formed, leading to higher with methoxyl at δH = 3.84 ppm (δ C = 55.6 ppm), this absorbance values. The high antioxidant activity of phenolic supposes that this methoxyl group is carried by the C-4'carbon. substances is often attributed to their -OH moieties (18). These Two correlation spots with a carbon atom at δ = 148.8 ppm are potent H donors because they allow the delocalization of attributable to C-2. The H-5'proton shows two correlation electrons across the molecule. This fact can explain why this spots: The first with the carbon atom at δ = 125.0 ppm isoflavonoid has a weak antioxidant activity, because some - attributable to C-1' and the second with the carbon atom at δ = OH groups are methoxylated. We evaluated antiplasmodial 146.6 ppm attributable to C-3'. This attribution is confirmed by activity against the chloroquine-sensitive Plasmodium the correlation task of this ring (C-3') and H-2'. Oxygenated falciparum 3D7 strain for compound 1 and EA extract. In carbon C-7 at δ = 161.8 ppm shows three correlation spots: The conclusion, one new compound was isolated from the ethyl first with proton H-8 at δ = 6.28 ppm, the second with proton acetate root extract of Allexis obanensis. The antibacterial, H-6 at δ = 6.42 ppm and the third with protons of the methoxyl antioxidant and antiplasmodial activities of this compound group δ = 3.9 ppm, which predicts the position of the methoxy isolated were assessed in this study. Compound 1 (IC50 group on the C-5 carbon. This is confirmed by the correlation =12.47±1.44 µM) showed good antiplasmodial activity against between proton H-6 at δ = 7.9 ppm and oxygenated carbon C- the chloroquine-sensitive Plasmodium falciparum 3D7 strain 5. Hence the structure: but has presented a weak antioxidant activity.It also presented good activities against many bacterial strains. The Antimicrobial results: The results of antibacterial and antiplasmodial properties of compound 1 support the ethno- antifungal activities of EA extract and compound 1 from medicinal use of Allexis obanensis roots in the treatment of Allexisobanensis are presented in tables 1, 2, 3 and 4.The malaria and stomach aches. activity of ethyl acetate was not very different from that of compound 1. Nine strains of Gram-negative bacteria including Experimental reference strains (ATCC) and multi-resistant clinical isolates were used. These strains belonging to different bacterial General experimental procedures: Melting points were species are distributed as follows: determined on an Electro thermal I A 9000 series digital A strain of Escherichia coli (ATCC8739). melting point apparatus and were uncorrected. The UV spectra A strain and three clinical isolates of Enterobacter were recorded on UV-570/VIS/NIP and Shimadzu UV- aerogenes (ATCC13048; CM64; EA289; EA294). 24012A double-beam spectrophotometers. IR measurements were obtained on a PerkinElmer (model 1600) FTIR Two clinical isolates of Enterobacter cloacae 1 13 (BM67,K2). spectrometer. The 1D ( H, C, DEPT) and 2D (COSY, NOESY, HSQC and HMBC) NMR spectra were recorded in A clinical isolate from Providencia stuartii DMSO-d6 and MeOH-d4 using a Bruker 600 (600 MHz for (PS299645). 1H NMR, 150 MHz for 13C NMR) spectrometer. ESIMS were A strain of Klebsiella pneumoniae (ATCC11296). obtained using an MSQ Thermofinnigan instrument. Chemical shifts are stated in parts per million (ppm) from The works of Berche (1993), Fauchère and Avril (2002), tetramethylsilane (TMS) internal standard. Flash column showed that when the CMB of an antibiotic on a given strain is chromatography was performed using silica gel 60 (Merck, close to the MIC (CMB/MIC = 1 or 2), the antibiotic is said to 0.040–0.063 mm). TLC was conducted on pre-coated Merck be bactericidal, in contrast, if these values are relatively distant Kieselgel 60 F254 plates (20×20 cm, 0.25 mm). Spots were (4
Plant material: Allexis obanensis was collected on 7th June Extraction and isolation: Dried and powdered root of Allexis 2014 at Bidou II, 20 km from the town of Kribi (South obanensis (1 kg) was extracted with MeOH (3L) at room Cameroon) under the leadership of Mr. NANA (Botanist). temperature and evaporated under vacuum to yield a crude HO extract (200g). O
OH
OMe O
OMe Compound 1. 7-hydroxy-3-(3-hydroxy-4-méthoxyphenyl) -5-méthoxy-4H-chromen-4-one
Table 1. MIC of EA extract and compound 1 on yeast strains (mg/mL)
Reference Microorganism EA extract 1 Fluconazole Ampicillin C. albicans >0.5 >0.5 0.032 C. krusei >0.5 >0.5 0.032 C. parapsilosis >0.5 >0.5 0.032 S. aureus NR46374 >0.5 >0.5 0.000488 K. pneumonia NR41916 >0.5 >0.5 0.000488 S. enterica NR13555 >0.5 >0.5 0.000488
Table 2. MFC of EA extract and compound 1 on yeast strains (mg/mL)
Reference Microorganism EA extract 1 Fluconazole Ampicillin C. albicans >0.5 0.5 0.032 C. krusei >0.5 0.25 0.032 C. parapsilosis >0.5 >0.5 0.032 S. aureus NR46374 >0.5 >0.5 0.000488 K.pneumonia NR41916 >0.5 >0.5 0.000488 S. enterica NR13555 >0.5 >0.5 0.000488
Table 3. Antibacterial activities
Compounds CM64 BM67 ATCC 8739 K2 PS299 645 ATCC 13048 EA289 ATCC 11296 EA294 EA extract 2 ND ND 1 ND 2 2 2 ND 1 2 2 2 ND 2 ND 2 2 2
Table 4. Antioxidant Activities
Radical scavenging activity Reducing power SC50(µM/L) EC50(µM/L) FRAPµg EAA/mgdw 1 07.50±0.48 192.50 129.45±3.5421 AA 04.50±0.28 112.50 206.48±12.33
Table 5. Antiplasmodial activities
Compounds IC50 (3D7 strain P. falciparum ) 1 12.47±1.44 µM EA 0.90±1.21 µM Chloroquine 0.006±0.3 µM
The identification was carried out at the National Herbarium of 100g of this extract was dissolved in MeOH-H2O (8:2) and Cameroon by Mr. NANA in comparison with specimen partitioned with n-hexane (3×150 mL) and ethyl acetate number 31839/HNC. Identified in Gabon and southern (3×200 mL). The ethyl acetate portion (35g) was subjected to Cameroon (Kribi) in the Kienke forest, it is a small Shrub up to column chromatography over silica gel eluting with gradients 6 m tall, with a pale brown smooth stem and small leaves. The of CH2Cl2/MeOH to produce 79 fractions of 250 mL each. flowering is done on the stem. It has pedicel 15mm long These fractions were combined based on their TLC profiles (Achoundong and Onana, 1998). into 3 major fractions: A (5.2 g,1–40); B (3.6g, 41–55); C (3g, 56–79). Fractions A (CH2Cl2/MeOH50:1); B(CH2Cl2/MeOH 40:1); C (CH2Cl2/MeOH 30:1). 72882 Nganso Ditchou Yves Oscar et al. New isoflavonoid from the roots of allexis obanensis (violaceae) and evaluation of antibacterial, antioxidant and antiplasmodial activities
Fraction A (CH2Cl2/MeOH 50:1) was purified by silica gel A 2.5 mL aliquot of the supernatant was mixed with 2.5 mL of column chromatography with a gradient of CH2Cl2/EtOAC ultra-pure water and 0.5 mL of ferric chloride (0.1%). The (20:1) to yield compounds 1(18mg). absorbance of this mixture was measured at 700 nm. A greater absorbance value indicates greater reducing power. Each In vitro evaluation of the antibacterial activity of the crude solution was analysed in triplicate, and the average values were 3+ extracts: 7.6 g of Mueller Hinton Agar (MHA) was dissolved plotted to obtain the IC50 of Fe reduction by linear regression. in 200 mL of distilled water and then heated on autoclave at The activity of an ascorbic acid solution was used for 121 °C for 30 min. After cooling the mixture was poured into normalisation. the petri dishes near the beak of Bunsen burner. In vitro antiplasmodial assays: The culture of P. falciparum Liquid medium: 13.65 g of Mueller Hinton Broth (MHB) and antiplasmodial test were carried out as previously were dissolved in 650 mL of distilled water. A part of this described (Frédérich, 2001).The asexual erythrocytic stage of medium was distributed in tubes of 15mL (10.853mL per tube P. falciparum chloroquine-sensitive 3D7 strain (from Prof. which will be used for inocula). Another part was distributed Grellier of Museum d’Histoire Naturelle, Paris, France) was in the 2 mL tubes (1.7 mL per tube for the dilution of the cultivated continuously in vitro according to the procedure extracts). These tubes and the rest of medium were heated in described by Trager and Jensen (1976) under an atmosphere of an autoclave at 121°C for 30 min. 5% CO2, 5% O2 and 90% N2 at 37 °C. The host cells were human red blood cells (A or Rh+). The culture medium was Culture of bacterial strains: The different bacterial strains RPMI 1640 (Gibco) containing 32 mM NaHCO3, 25 mM were subcultured by the method of the streaks on MHA agar HEPES and 2.05 mM L-glutamine. The medium was medium poured into the Petri dishes. The petri dishes were supplemented with 1.76 g/L of glucose (Sigma-Aldrich), 44 introduced into the incubator at 37 °C. for 18 hours in order to mg/mL of hypoxanthin (Sigma-Aldrich), 100 mg/L of obtain a young culture and isolated colonies. The isolated gentamycin (Gibco), and 10% human pooled serum (A or O colonies were used to prepare the inoculum. Rh+). Parasites were subcultured every 3–4 days with initial conditions of 0.5% parasitaemia and 1% hematocrit. The Preparation of the inoculums: Using a sterile platinum loop, EtOAC extract and pure compound were evaluated in vitrofor a few colonies of bacteria from each strain were taken from the their activity against P. falciparum(3D7). Artemisinin (98%, activation medium and each introduced into a tube containing Sigma-Aldrich) was used as standard (IC50 4.12 ng/mL). First, a sterile physiological solution (0.9% NaCl). The contents of stock solutions of extract and pure compound were prepared in each tube were homogenized using the vortex in order to DMSO at a concentration of 20 mg/mL. The solutions were obtain a turbidity comparable to the standard scale of Mc further diluted in media to give 2 mg/mL stock solutions. The Farland (Table 1) corresponding to the concentration of 1.5. highest concentration of solvent to which the parasites were 108CFU/mL. Subsequently, 147 μL of the resulting suspension exposed was 1%, which was shown to have no measurable was removed and introduced into 10.85 mL of MHB for a effect on parasite viability. All tests were performed in 6 volume of 11000 mL of an inoculated medium at 2.10 triplicate. The results are expressed as the mean IC50 (the CFU/mL. concentration of a drug that reduced the level of parasitaemiato 50 %). Evaluation of antioxidant activity and antiplasmodial activity Acknowledgement
DPPH radical-scavenging activity: The ability of compounds All thanks to the Financial support from the «Institut 1, and 3–7 to scavenge DPPH free radicals was evaluated Agricoled’Obala», Mr Nkoa Alima and Ndjie Louisfor all according to the method of Brand-williams et al.(1995).4 mL material support. aliquot of the sample solution was mixed with 1 mL of DPPH (0.04 mM in methanol). This mixture was vigorously shaken at REFERENCES room temperature for 30 min. The absorbance of the mixture was then measured at 515 nm. A low absorbance value Abdullahi, M.I., LLiya, L., Haruna, A.K., Sule, M.I., indicates effective free radical scavenging. Each solution was Abdullahi, M.S. 2010. Preliminary Phytochemical and analysed in triplicate, and the average values were plotted to Antimicrobial investigations of leaf extracts of obtain the SC50 against DPPH by linear regression. The Ochnaschweinfurthiana (Ochnaceae). Afr. J. Pharm. activity of ascorbic acid, a recognised antioxidant, was used as Pharmacol. 4: 083–086. a standard over the same range of concentrations. The radical Abdullahi, M.I., Musa, A.M., Haruna, A.K., Sule, M.I., scavenging activity was evaluated as the percentage of Abdulmalik, I.A., Abdullahi, M.S., Abimiku, A.G., LLiya, inhibition according to the following equation: L. 2011. Anti-microbial flavonoid diglycoside from the
leaves of Ochnaschweinfurthiana Hoffm (Ochnaceae). Niger. J. Pharm. Sci., 10: 1–7. Achoundong et Onana J.M. 1998. Allexiszygomorpha Evaluation of reducing power: The reducing powers of (Violaceae): a new species from the littoral forest of compounds 1 was evaluated according to the method of Benzie Cameroon. Kew Bulletin, 53 (4):1009-1010. and Strain (1999). 25 mL test tube was loaded with 1.0 mL of Achoundong, G. 2010. The African genus Allexis (Violaceae). sample solution, 2.5 mL of phosphate buffer (2 M, pH 6.6) and A synoptic revision, National Herbarium,p9. 2.5 mL of 1% (m/v) K3(Fe(CN)6). The mixture was incubated Akoua, K.C., Guessend, N., Gbonon, V., Faye-kette A.Y.H., at 45 °C for 20 min. Next, 2.5 mL of trichloroacetic acid (10% Dosso, M. 2004. Methicillini-resistant of S. aureus activity m/v) was added, and the solution was centrifuged at 4000 rpm in Abidjan A new hospital problem. Medecines Maladies for 15 min. Infectieuses, (1998-2001),34(3): 132-36. 72883 International Journal of Current Research, Vol. 10, Issue, 08, pp.72879-72883, August, 2018
Aligiannis, N., Kalpotzakis, E., Mitaku, S., Chinou, I.B. 2001. Messi, A.N., Ngo M.J., Ndongo, J.T., Nyegue, M.A., Tiabou Composition and antimicrobial activity of the essential oils T.A., Ladoh, Y.F., Michel, F., Pegnyemb, D.E. 2016. of two Origanum species. Journal of Agricultural and Phenolic compounds from the roots of Food Chemistry, 40: 4168-4170. Ochnaschweinfurthiana and their antioxidant and Arabski, M., Węgierek-Ciuk A., Czerwonka G., Lankoff, A., et antiplasmodial activities, Phytochemistry Letters. 2016, 17: Kaca, W. 2012. Effects of Saponins against Clinical E. coli 119–125. Strains and Eukaryotic Cell Line. Journal of Biomedicine Moon, H.I., Jung, J.C., Lee, J. 2007. Antiplasmodial activity of and Biotechnology, 6: 3. triterpenoid isolated from whole plants of Viola genus from Feng, S., Hao, J., Xu, Z., Chen, T., Qiu, S.X.S. 2012. South Korea.Parasitol. Res., 100: 641–644. Polyprenylatedisoflavanone and isoflavonoids from Moon, H.I., Jung. J.C., Lee. J. 2007. Antiplasmodial activity of Ormosiahenryiand their cytotoxicity and anti-oxidation triterpenoid isolated from whole plants of Viola genus from activity. Fitoterapia, 83: 161–165. South Korea. Parasitol Res., 100: 641–644. Frédérich, M., De Pauw, M.C., Prosperi, C. 2001. Nganso, Y.O.D., Ngantchou, I.E.W., Nkwenoua, E., Nyasse, Strychnogucines A and B, two new B., Denier, C., Hannert, V., Schneider, B. 2011. antiplasmodialbisindole alkaloids from Strychnosicaja.J. Antitrypanosomal and Cytotoxic Activities of 22- Nat. Prod., 64: 12–16. Hydroxyclerosterol, a New Sterol from Allexiscauliflora Ghogomu, R., Sondengam, B.L., Martin, M.T., Bodo, B., (Violaceae), Sci Pharm., 79: 137–144. Lophirone A. 1987. Abiflavonoid with unusual skeleton Vukics, V., Kery, A., Bonn, G.K., Guttman, A. 2008. Major from Lophiralanceolata. Tetrahedron Lett. 28: 2967–2968. flavonoid components of heartsease (Viola tricolor L.) and Gülçin, I., Alici, H.A., Cesur, M. 2005. Determination of in their antioxidant activities. Anal Bioanal Chem., 390: vitro antioxidant and radical scavenging activities of 1917–1925. propofol. Chem. Pharm. Bull. 53: 281–285. Hutchinson, J., Dalziel, J.M. 1954. Crown Agents for oversea Governments and Administrations. Millbank, London.
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